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• • C—CHEMISTRY; METALLURGY
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Definitions

• the present invention relates to aqueous pharmaceutical compositions stabilized with meglumine for long-term storage of etanereept, methods of manufacture of the compositions, methods of their administration, and kits containing the same.
• the invention includes etanereept formulations that do not require arginine for stabilization.
• Polypeptides must often be stored prior to their use. When stored for extended periods, polypeptides are frequently unstable in solution (Manning et .al., 1989, Pharm. Res. 6:903-918). To extend their shelf life, additional processing steps have been developed, such as drying, e.g., lyophilization. However, !yophilized pharmaceutical compositions are less convenient to use.
• Typical practices to improve polypeptide stability can be addressed by varying the concentration of elements with the formulation, or by adding excipients to modify the formulation (See, for example, U.S. Pat. Nos. 5,580,858 and 6,171 ,586).
• the use of additives can still result in inactive polypeptides, in addition, in the case of lyophilization, the rehydration step can result in inactivation of the polypeptide by, for example, aggregation or denaturation (Hora et a!., 1992, Pharm. Res., 9:33-36; Liu et al., 1991 , Biotechnoi. Bioeng., 37:177-184).
• Another way to improve polypeptide stability is to use L-arginine at a specific concentration ⁇ U.S. Pat. No. 7,648,702).
• etanereept (Enbrel ⁇ , immunex Corporation), which is a dimeric fusion polypeptide consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgGl it consists of 934 amino acids and has an apparent molecular weight of approximately 150 kiioda!ions (Physicians Desk Reference, 2002, Medical Economics Company Inc.)
• the Fc component of etanercept contains the constant heavy 2 (CH2) domain, the constant heavy 3 (CH3) domain and hinge region, but not the constant heavy 1 (CM) domain of human igG1.
• An Fc domain can contain one or ali of the domains described above.
• Etanercept is usually produced by recombinant DMA technology in a Chinese hamster ovary (CHO) mammalian cell expression system.
• the present invention provides novel stable liquid formulations of etanercept that allow its long-term storage.
• the present invention is an aqueous stabilized formulation of etanercept comprising: etanercept; and stabilizing ingredients to retard instability, aggregation and/or fragmentation of the etanercept in the formulation, said stabilizing ingredients being comprised of at least one of the following: (a) meglumine; or (b) meglumine in combination with sucrose; or (c) meglumine in combination with sodium chloride; or ⁇ (d) meglumine in combination with sodium chioride and sucrose.
• the stabilized etanercept formulations of the present invention elicit long term storage stability as characterized by at least one of: (1) SEC analysis at M 3 or T 2 or T 4 of: monomer content greater than about 90%: aggregates content of less than about 3 wt%; and fragment 3 content less than about 5 wt%: and (2) H!C analysis at fv1 3 or T 2 , or T wherein the amount of the composition represented by peak 1 of the HIC chromatogram is less than about 3 wt.%; the amount of the composition represented by peak 2 of the HIC chromatogram is greater than 80 wt.%; and the amount of the composition represented by peak 3 of the HIC chromatogram is less than about 20 wt.%.
• the formulations elicit long term storage stability as characterized by: an HIC analysis at M 3 or T 2 or T 4 wherein the amount of the composition represented by peak 2 of the HIC chromatogram is greater than or equal to about 95 wt.%; and wherein, if peak 3 is present on the HIC chromatogram, the amount of the composition represented by peak 3 is less than or equal to about 3 wt.%.
• the stabilized etanercept formulation as summarized above optionally and preferably, contains no arginine, or are essentially free of arginine.
• the formulations of the invention have excellent stability as determined by SEC (Size Exclusion Chromatography) and HIC (Hydrophobic Interaction Chromatography) analysis conducted after one, two or three months of storage at 5° C. These formulations are comparable to or better than a commercially available formulation of etanercept, in which arginine is a required component.
• the present invention is further directed to formulations of stabilized etanercept, as summarized above, which contain no arginine, or are essentially free of arginine, and wherein the composition, evaluated at M 3 or T 2 or T 4 , elicits long term storage stability that meets one or both of the following criteria: (A) stability comparable to or better than commercially available etanercept marketed under the trademark Enbrel®, as measured by (i) SEC analysis of the amounts of aggregate(s), monomer and fragment 3 in the composition (as defined in the specification) and (ii) HIC analysis of amounts of material in the composition corresponding to peaks 1 , 2 and 3 of the HIC chromatogram (as defined in the specification); and (B) an HIC chromatogram in which (i) peak 3 is absent, or essentially absent and (ii) peak 2 represents greater than about 95 wt% of the composition; an SEC chromatogram containing essentially no peak corresponding to aggregate(s); and an SEC chromatogram in which
• the formulation of the invention comprises about about 25 to 75 mg/ml etanercept, sodium chloride in an amount up to about 150 mM, about 1 to about 30 mM sodium phosphate; and about 0 to 5 wt.% sucrose or trehalose or combination thereof; wherein the composition has a pH of about 6.0 to about 6.6; and wherein the composition is characterized by SEC analysis at 3 or T 2 or T 4 in which: monomer content is greater than about 85 wt.%; aggregate(s) content is less than about 3 wt.%, and fragment 3 content is less than about 8 wt.%. Formulations meeting these analytical criteria do not require arginine for stabilization.
• the stabilized etanercept formulation is further characterized by (a) an SEC analysis at 3 or T 2 or T 4 of greater than about 90 t% monomer content; and less than about 3 wt% aggregate(s) content; and (b) an HIC analysis at M 3 or T 2 or T 4 wherein the amount of the composition represented by peak 1 of the HIC chromatogram is less than about 4 wt%; the amount of the composition represented by peak 2 of the HIC chromatogram is greater than about 80 wt%; and the amount of the composition represented by peak 3 of the HIC chromatogram is less than about 20 wt%; and wherein the formulation is free or essentially free of arginine.
• the etanercept compositions of the invention further afford the ability to provide formulations which contain acceptable levels of subvisible particles. Accordingly, the invention is further directed to stabilized etanercept formulations having, at M 3 or T 2 or T 4l no more than, on average, about 10,000 subvisible particles per mL having a size greater than 5 pm.
• the stabilized etanercept composition of the present invention are further characterized by: (a) an SEC analysis at IV or T 2 or T of greater than about 90 wt% monomer content; and less than about 3 wt.% aggregate(s) content; and (b) an HIC analysis at 3 or T 2 or T 4 wherein the amount of the composition represented by peak 1 of the HIC chromatogram is less than about 3 wt%; the amount of the composition represented by peak 2 of the HIC chromatogram is greater than 80 wt%; and the amount of the composition represented by peak 3 of the HIC chromatogram is less than about 20 wt% and wherein the formulation is free or essentially free of arginine.
• the stability of the formulations may be further characterized in that the compositions, optionally free or essentiaily of arginine, exhibit an HIC analysis at 3 or T ⁇ or T 4 wherein the amount of the composition represented by peak 1 of the HIC chromatogram is less than about 1%; the amount of the composition represented by peak 2 of the HIC chromatogram is greater than about 95 t%; and the amount of the composition represented by peak 3 of the HIC chromatogram is less than about 1 wt%.
• Formulations meeting these analytical criteria do not require arginine for stabilization.
• Preferred stabilized compositions of the invention, preferably free or essentlaliy free of arginine exhibit an HiC analysis at M 3 or T ?
• the amount of the composition represented by peak 1 of the HIC chromatogram is less than about 2% or preferably less than about 1%; the amount of the composition represented by peak 2 of the HIC chromatogram is greater than about 95 wt.% and preferably greater than about 97%; and the amount of the composition represented by peak 3 of the HIC chromatogram is less than about 1 wt% : and preferably 0 to 1%,
• nstability denotes the tendency of the etanercept monomer to undergo a variety of undesired transformations during storage.
• transformations include the formation of oligomers and high molecular weight aggregate(s) (hereinafter terms “aggregate(s)” in which multiple copies of the essentially intact etanercept monomer become irreversibl associated with one another through a variety of non-covalent attractions (e.g., electrostatic interactions.)
• Undesired transformations during storage may also include degradation of the etanercept monomer to smaller fragments and/or clipped species.
• a formulation of etanercept should minimize, to the greatest extent possible, the tendency of the formulation to result, during storage, in the formation of aggregates, misfolded protein, oligomers and/or fragments of etanercept.
• An important benefit resulting from the ability to reduce formation of unwanted aggregates or fragments is a reduction in the potential toxicity and/or immunogenicity of the drug.
• the etanercept formulation of the present invention which is optionally and preferably free, or essentially free of argihine.
• arginine is intended to mean that arginine, even if present, is not contributing to the stabilization of the etanercept monomer in the formulation to such an extent that a person skilled in the art would judge its presence beneficial or necessary from a stabilization standpoint.
• etanercepf or "etanercept monomer” or “monomer” is synonymous with Enbre!®. It refers to a polypeptide which is a dimeric fusion polypeptide consisting of the extracellular iigand-binding portion of the human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgGl It consists of 934 amino acids and has an apparent molecular weight of approximately 150 kiiodaStons.
• etanercepf also encompasses etanercepf with minor modifications in the amino acid structure (including deletions, additions, and/or substitutions of amino acids) which do not significantly affect the function, potency, or avidity of etanercepf.
• etanercepf encompasses ail forms and formulations of Enbrel®, including but not limited to concentrated formulations, injectable ready-to-use formulations; formulations reconstituted with water, alcohol, and/or other ingredients, and others.
• sugar refers to monosaccharides, disachharides, and polysaccharides.
• sugars include, but are not limited to, sucrose, trehalose, dextrose, and others.
• meglumine refers to a compound with chemical formula H 3 NHCH2(CHOH) 4 CH 2 OH, also known as 1-Deoxy ⁇ 1-methylamtnosorbitol; N- ethyl- d-giucamine; and 1-Deoxy-1-methylamino-D-glucitol.
• polyo refers to an alcohol containing multiple hydroxy! groups.
• examples of polyois include, but are not limited to, mannitoi, sorbitol, and others.
• long-term storage is understood to mean that the pharmaceutical composition can be stored for three months or more, for six months or more, and preferably for one year or more. Long-term storage is also understood to mean that the pharmaceutical composition is stored either as a liquid at 2-8° C, or is frozen, e.g., at -2G°C, or colder. It is also contemplated thai the composition can be frozen and thawed more than once.
• stable or “stabilized” with respect to long-term storage is understood to mean that etanercepf contained in the pharmaceutical compositions does not lose more than 20%, or more preferably 15%, or even more preferably 10%, and most preferably 5% of its activity relative to activity of the composition at the beginning of storage,
• mammal includes, but is not limited to, a human.
• pharmaceutically acceptable carrier refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material, formulation auxiliary, or excipient of an conventional type.
• a pharmaceutically acceptable carrier is non- toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
• composition refers to a mixture that usually contains a carrier, such as a pharmaceutically acceptable carrier or excipient that is conventional in the art and which is suitable for administration into a subject for therapeutic, diagnostic, or prophylactic purposes. It may include a ceil culture in which the polypeptide or polynucleotide is present in the cells or in the culture medium.
• a carrier such as a pharmaceutically acceptable carrier or excipient that is conventional in the art and which is suitable for administration into a subject for therapeutic, diagnostic, or prophylactic purposes. It may include a ceil culture in which the polypeptide or polynucleotide is present in the cells or in the culture medium.
• compositions for oral administration can form solutions, suspensions, tablets, pills, capsules, sustained release formulations, oral rinses or powders.
• treatment refers to any administration or application of remedies for disease in a mamma! and inciudes inhibiting the disease, arresting its development, relieving the disease, for example, by causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process.
• treatment includes obtaining a desired pharmacologic and/or physiologic effect, covering any treatment of a pathological condition or disorder in a mammal.
• the effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse affect attributable to the disorder.
• disease refers to any condition, infection, disorder or syndrome that requires medical intervention or for which medical intervention is desirable. Such medical intervention can include treatment, diagnosis and/or prevention.
• an effective amount of the polypeptide of the invention for administration to the living subject is an amount that prevents and/or treats an integrin av 3-mediated disease.
• the exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
• Ti refers to a point in time at which an etanercept formulation has been stored for about one week at 40° C
• T 2 refers to a point in time at which an etanercept formulation has been stored for about two weeks at 40° C
• M3 refers, collectively, to three points in time, and in particular to an analytical result being observed for an etanercept formulation after duration of either about one, about two or about three months of storage at a storage temperature of 5° C.
• reference herein to an analysis being conducted at M3 should be understand to mean that such analysis is be done at the point in time at which etanercept formulation has been in storage for a duration selected from about one, about two, or about three months.
• an etanercept formulation elicit a certain analytical value or measurement at 3 is satisfied if the required value is observed at a point in time corresponding to at least one of the following storage durations: at approximately one month, at approximately two months or at approximately three months of storage at 5°C.
• Peak 1 Peak 2
• Peak 3 Peak 3 when used herein in connection with discussion of HIC chromatography results refers to the same peaks 1 , 2 and 3 discussed in US Patent 7,294,481.
• the present invention provides several embodinients of aqueous formulations of etanercept that allow stable long-term storage of etanercept, so that etanercept is stable over the course of storage either in liquid or frozen states.
• the provided formulations include, but are not limited to formulations that do not contain arginine and do not require any extra steps such as rehydratlng. These embodiments are explained in a greater detail below.
• compositions of the present invention comprise etanercept (Enbrel®).
• etanercept is a dimeric fusion poiypeptide consisting of the extracellular Iigand-binding portion of the human 75 ki!odalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human igG .
• TNFR tumor necrosis factor receptor
• Etanercept consists of 934 amino acids.
• the Fc component of etanercept contains the constant heavy 2 (CH2) domain, the constant heavy 3 (CH3) domain and hinge region of human IgG
• An Fc domain can contain one or all of the domains described above.
• Etanercept suitable for storage in the present pharmaceutical composition can be produced by living host ceils that express etanercept, such as hybridomas in the case of antibodies, or host celis that that have been genetically engineered to produce the polypeptide in the case of fusion polypeptides or antibodies.
• Methods of genetically engineering cells to produce polypeptides are well known in the art. See, e.g., Ausubei et a!., eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods include introducing nucleic acids that encode and aliow expression of the polypeptide Into living host ceils. These host cells can be bacterial cells, fungal cells, or, preferably, animal ceils grown in culture.
• Bacterial host ceils include, but are not limited to, Escherichia coii cells. Examples of suitable £ coii strains include: HB101 , DH5.alpha, GM2929, JM109, KW251 , NM538, N 539, and any E. coii strain that fails to cleave foreign DNA.
• Fungal host cells that can be used include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus ceils,
• a few examples of animal cell lines that can be used are CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, and W138. New animal cell lines can be established using methods weli know by those skiiied in the art (e.g., by transformation, viral infection, and/or selection).
• etanercept can be secreted by the host celis into the medium.
• etanercept Purification of the expressed etanercept can be performed by any standard method. When etanercept is produced intracellular ⁇ , the particulate debris is removed, for example, by centrlfugation or ultrafiltration. When etanercept is secreted into the medium, supernatants from such expression systems can be first concentrated using standard polypeptide concentration filters. Protease inhibitors can also be added to inhibit proteolysis and antibiotics can be included to prevent the growth of microorganisms.
• Etanercept can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and any combination of known or yet to be discovered purification techniques, including but not limited to Protein A chromatography, fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSET®, an anion or cation exchange resin chromatography (such as a polyaspartic acid column), chromatofocusing, SDS- PAGE, and ammonium sulfate precipitation.
• Protein A chromatography fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSET®, an anion or cation exchange resin chromatography (such as a polyaspartic acid column), chromatofocusing, SDS- PAGE, and ammonium sulf
• the invention provides a stable aqueous formulation comprising etanercept and an ionic poiyol derivative excipient, wherein said excipient is selected from the group consisting of meglumine (N-methyi-D-glucamine), mannosylg!ycerate, glucosylglycerate, mannosyllactate, mannosylglycolate, and diglycerolphosphate.
• excipient is selected from the group consisting of meglumine (N-methyi-D-glucamine), mannosylg!ycerate, glucosylglycerate, mannosyllactate, mannosylglycolate, and diglycerolphosphate.
• the invention is an aqueous stabilized formulation of etanercept comprising: etanercept; and stabilizing ingredients to retard instability, aggregation and fragmentation of the etanercept in the formulation, said stabilizing ingredients being comprised of (a) meglumine; or (b) meglumine in combination with sucrose; or (c) meglumine in combination with sodium chloride; or (d) meglumine ' in combination with sodium chloride and sucrose.
• Meglumine is commonl used as a small molecule excipient
• meglumine is also able to stabilize aqueous pharmaceutical compositions containing a large protein, such as etanercept.
• meglumine reduces etanercepfs tendency to associate in undesired ternary or quaternary complexes, and therefore, reduces aggregation of etanercept
• the reduction in aggregation is believed to last for a long period of time, e.g., two years or more.
• ionic poiyol derivatives, including meglumine are able to stabilize aqueous pharmaceutical compositions containing etanercept by a combination of three different mechanisms. First, they can act as an excluded solute in the same way mannitol, sucrose, and sorbitol increase conformational stability.
• charged solutes can alter the colloidal stability, thereby reducing the propensity to self-associate, thereby slowing aggregation.
• these ionic polyoS derivatives being charged near neutral H, can act as salting-in agents, as arginine does, potentially resolubilizing aggregates.
• the stabilizing effects of meglumine are not limited to reduction in aggregates but may involve other aspects of stabiiization of the etanercept monomer in a formulation containing the monomer.
• compositions of the invention may be prepared by combining, a purified etanercept and the ionic polyol derivative, preferably meglumine, Further, a buffer, a tonicity modifier and an additional excipient and other commonly used inactive ingredients can be added as needed. For simplicity, these are discussed more fully later in the specification.
• a buffer can be added first, middle or last
• the tonicity modifier can also be added first, middle or last.
• some of these chemicals can be incompatible in certain combinations, and accordingly, are easily substituted with different chemicals that have similar properties but are compatible in the relevant mixture.
• the concentration of meglumine in the provided formulations is preferably between about 0.1% ⁇ w/v) to 40% (w/v), more preferably about 1% to about 20%, more preferably about 2% to about 10%, even more preferably about 2% to about 5%.
• Meglumine is available from commercial suppliers.
• a preferred embodiment comprises about 25 to about 75 mg/ml etanercept, about 1-30 mM of sodium phosphate; up to about 10% meglumine; optionally up to about 5 wt.% sucrose; and optionally up to about 100 mM sodium chloride, wherein the composition has a pH of about 6.0 to 7.0, and preferably about 6.0 to about 6.6 and most preferably about 6.3 to about 6.5.
• a meglumine stabilized etanercept composition is preferably characterized by SEC analysis at T 2 in which: the monomer content is greater than about 85 wt.%; aggregate(s) content is less than about 3 wt.%; and fragment 3 content is Sess than about 8 wt.%.
• a particularly preferred etanercept formulation stabilized with meglumine is characterized by HIC analysis at T 4 or T 2 wherein the amount of the composition represented by peak 1 of the H!C chromatogram is less than about 1%; the amount of the composition represented by peak 2 of the H!C chromatogram is greater than about 99 wt.%; and the amount of the composition represented by peak 3 of the HIC chromatogram is less than about 1 wt.%.
• meglumine can be replaced with another sonic po!yol derivative of sorbitol, glycerol or mannito!, such as mannosylgiycerate, glucosylglycerate, mannosyllactate, mannosyiglycolate, and diglyceroiphosphate (at about 0.1% to about 40%) in the formulation.
• a preferred meg!umine-siabiiized etanercept formulation free of arginine and exhibiting analytical properties as described above comprises about 25 to about 75 mg/ml etanercept; about 0,5 wt.% megiumine; about 25 mM phosphate; about 1% sucrose; and about 100 mM sodium chloride.
• a further preferred meglumine-stabilized etanercept formulation free of arginine and exhibiting analytical properties as described above comprises about 50 mg/ml etanercept; about 5 wt.% meglumine; about 25 mM phosphate.
• the etanercept formulations comprising ionic poiyol derivatives such as meglumine for stabilization according to the present invention are preferably free or essentially free of arginine.
• the formulations of the invention may also include buffers, tonicity modifiers, excipients, pharmaceutically acceptable carriers and other commonly used inactive ingredients of the pharmaceutical compositions. For simplicity, these are discussed more fully later in the application.
• Buffers maintain p ' H in a desired range.
• Suitable buffers include histidine, potassium phosphate, sodium or potassium citrate, maleic acid, ammonium acetate, tris-(hydroxymethyi)-aminomethane (tris), various forms of acetate and diethanolamine.
• concentration of the buffer in the formulation is preferably between about 1 mM to about 1M, and more preferably about 10 mM to about 200 mM. Buffers are well known in the art and are manufactured by known methods and available from commercial suppliers.
• buffers examples include phosphate, histidine, citrate, maleate, tartrate, succinate, acetate, tris-(hydroxymethyl)-aminomethane (tris), bicarbonate.
• the buffer is sodium phosphate.
• the pH of the pharmaceutical composition is at or near physiological levels.
• the pH of the provided compositions Is between about 5.8 and about 8.4; and even more preferably, betwee about 6.2 and about 7.4.
• the pH can be adjusted as necessary to maximize stabilit and solubility of etanercept in a particular formulation.
• etanercept formulations at a phi outside of physiological ranges, yet tolerable to the patient are also within the scope of the invention.
• a tonicity modifier is a molecule that contributes to the osmolality of a solution.
• the osmolality of a pharmaceutical composition is preferably adjusted to maximize the active ingredient's stability and/or to minimize discomfort to the patient upon administration. It is generally preferred that a pharmaceutical composition be isotonic with serum, i.e., having the same or similar osmolality, which is achieved by addition of a tonicity modifier.
• the osmolality of the provided formulations is from about 180 to about 420 mOs .
• the osmolality can be either higher or lower as specific conditions require.
• tonicity modifiers suitable for modifying osmolality include, but are not limited to amino acids (not including arginine) (e.g., cysteine, histidine and glycine), salts (e.g., sodium chloride, potassium chloride and sodium citrate) and/or saccharides (e.g., sucrose, glucose and mannitol).
• amino acids not including arginine
• cysteine e.g., cysteine, histidine and glycine
• salts e.g., sodium chloride, potassium chloride and sodium citrate
• saccharides e.g., sucrose, glucose and mannitol
• Preferred tonicity modifiers are glycine, alanine, sodium chloride, potassium chloride, and sodium sulfate.
• the concentratio of the tonicity modifier in the formulation is preferably between about 1 mM to about 1 , more preferably about 10 mM to about 200 mM.
• Tonicity modifiers are well known in the art and are manufactured by known methods and available from commercial suppliers.
• Excipients also referred to as chemical additives, co ⁇ solutes, or co-solvents, that stabilize the polypeptide while in solution (also in dried or frozen forms) can also be added to a pharmaceutical composition.
• Excipients are well known in the art and are manufactured by known methods and available from commercial suppliers.
• excipients include but are not limited to sugars/polyols such as: sucrose, lactose, glycerol, xylite!, sorbitol, mannitol, maltose, inositol, trehalose, glucose; polymers such as: serum albumin (bovine serum albumin (BSA), human SA or recombinant HA), dextran, poiy(viny alcohol) PVA, hydroxypropyl methylceliulose (HPfvIC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellu!ose (HEC); non-aqueous solvents such as: polyhydric alcohols, (e.g., PEG, and glycerol) and dlmethylformamide (D F); amino acids such as; proline, L-serine, sodium glutamic acid, alanine, glycine, lysine hydrochloride
• Preferred excipients are sucrose, lactose, glycerol, xyiitol, sorbitol, mannitoi, maltose, inositol, trehalose, glucose, bovine serum albumin (BSA), human serum albumin (HSA), recombinant albumin, dextran, PVA, hydroxypropyl methylcei!ulose (HP C), pol ethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethylceilulose (HEC), polyethylene glycol, ethylene glycol, glycerol, alanine, glycine, lysine hydrochloride, sarcosine, SDS, polysorbate 20, polysorbate 80, poioxamer 188, trimethyiamine N-oxide, betaine, zinc ions, calcium ions, magnesium ions, CHAPS, sucrose monolaurate, and 2-O-beta-manno
• concentration of one or more excipients in a formulation of the invention is/are preferably between about 0.001 to 5 weight percent, more preferably about 0.1 to 2 weight percent.
• the invention provides a method of treating a mammal comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to a mammal, wherein the mammal has a disease or disorder that can be beneficially treated with etanercept.
• the etanercept is derived from the same species of mammal as is to be treated with the composition.
• the mammal is a human.
• Diseases or disorders that can be treated with the provided compositions include but are not limited to rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener's disease (granulomatosis), Crohn's disease (or inflammatory bowel disease), chronic obstructive pulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia, psoriasis, and atopic dermatitis.
• Additional diseases or disorders that can be treated with the compositions of the present invention include those described in WO 00/62790, WO 01/62272, U.S. Patent Application No. 2001/0021380, and US Pat. 7,648,702 B2, the relevant portions of which are incorporated herein by reference.
• compositions may be administered to a subject in need of treatment by injection systemicaily, such as by intravenous injection; or by injection or application to the relevant site, such as by direct injection, or direct application to the site when the site is exposed in surgery; or by topical application.
• the invention provides a method of treatment and/or prevention of rheumatoid arthritis comprises administering to a mammal in need thereof a therapeutically effective amount of one of the provided etanercept compositions.
• the therapeutically effective amount of the etanercept in the provided compositions will depend on the condition to be treated, the severity of the condition, prior therapy, and the patient's clinical history and response to the therapeutic agent.
• the proper dose can be adjusted according to the judgment of the attending physician such that it can be administered to the patient one time or over a series of administrations.
• the effective etanercept amount per adult dose is from about 1-500 mg/m 2 , or from about 1-200 mg/m 2 , . or from about 1-40 mg/m 2 or about 5-25 mg/m 2 .
• a flat dose may be administered, whose amount may range from 2-500 mg/dose, 2-100 mg/dose or from about 10-80 mg/dose.
• an exemplary dose range is the same as the foregoing described dose ranges or lower and preferably administered two or more times per week at a per dose range of 25-100 mg/dose.
• an acceptable dose for administration by injection contains 80-100 mg/dose, or alternatively, containing 80 mg per dose.
• the dose can be administered weekly, biweekly, or separated by several weeks (for example 2 to 8).
• etanercept is administered at 25 to 75 mg/ml by a single subcutaneous (SC) injection.
• SC subcutaneous
• an improvement in a patient's condition will be obtained by administering a dose of up to about 100 mg of the pharmaceutical composition one to three times per week over a period of at least three weeks. Treatment for longer periods may be necessary to induce the desired degree of improvement. For incurable chronic conditions the regimen may be continued indefinitely. For pediatric patients (ages 4-17), a suitable regimen may involve administering a dose of 0.4 mg/kg to 5 mg/kg of etanercept, one or more times per week,
• the pharmaceutical formulations of the invention may be prepared in a bulk formulation, and as such, the components of the pharmaceutical composition are adjusted to be higher than would be required for administration and diluted appropriately prior to administration.
• the pharmaceutical compositions can be administered as a sole therapeutic or in combination with additional therapies as needed.
• the provided methods of treatment and/or prevention are used in combination with administering a therapeutically effective amount of another active agent.
• the other active agent may be administered before, during, or after administering the pharmaceutical compositions of the present invention.
• Another active agent may be administered either as a part of the provided compositions, or alternatively, as a separate formulation.
• Administration of the provided pharmaceutical compositions can be achieved in various ways, including parenteral, peroral, buccal, sublingual, nasal, rectal, intraperitoneal, intradermal, transdermal, subcutaneous, intravenous, intra-arterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal administration, intramuscular injection, intravitreal injection, and topical application.
• compositions of this invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly, intravenously, intraperitoneal, intracerebrospina!, Intra-articular, intrasynovial, intravitreal, and/or intrathecal.
• Parenteral administration can be by bolus injection or continuous infusion.
• Pharmaceutical compositions for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• compositions of the present invention are suitable for administration using these new methods, e.g., !nject-ease®, Genject®, injector pens such as GenPen® * and needleless devices such as ediJector® and BioJector®.
• the present pharmaceutical composition can also be adapted for yet to be discovered administration methods. See also Langer, 1990, Science, 249:1527-1533.
• compositions can also be formulated as a depot preparation.
• Such long acting formulations may be administered by implantation (for example subcutaneousiy or intramuscuiariy) or by intramuscuiar injection.
• the formulations may be modified with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• compositions may, if desired, be presented in a vial, pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient, in one embodiment the dispenser device can comprise a syringe having a single dose of the liquid formulation ready for injection.
• the syringe can be accompanied by instructions for administration.
• the present invention is directed to a kit or container, which contains an aqueous pharmaceutical composition of the invention.
• concentration of the polypeptide in the aqueous pharmaceutical composition can vary over a wide range, but is generally within the range of from about 0.05 to about 20,000 micrograms per milliliter (Mg/m! of aqueous formulation.
• the kit can also be accompanied by instructions for use.
• Etanercept formulations stabilized with meglumine may be prepared and tested using the procedures generally described below.
• Each solid formulation component is weighed to the amount required for a given volume of formulation buffer. These components are combined into a beaker or vessel capable of carrying and measuring the given volume of formulation buffer. A volume of deionized water equal to approximately 3 ⁇ 4 of the target given formulation buffer is added to the beaker, and the components are then soiublized. The pH of the buffer is adjusted to the target formulation pH using 1 M sodium hydroxide and/or 1 fvl hydrogen chloride. The final formulation buffer volume is then raised to the target volume through the addition of deionized water.
• Etanercept protein solution is placed in dialysis material housing (such as Thermo Scientific Slide-A-Lyzer MINI Dialysis Unit 10,000 MVVCQ), which is then placed in contact with the desired formulation buffer for 2 hours at 4°C.
• Formulation buffer volume to protein solution volume ratio should be no less than 1000:1.
• the dialysis housing and protein solution it contains is then placed in a second, equal volume of formulation buffer for an additional 12 hours at 4°C.
• Resulting protein solution is removed from the dialysis material housing, and the concentration of protein determined using ultraviolet spectroscopy. Protein concentration is adjusted to the desired level using centrifugation (such as Arnicon Ultra 10,000 MWCO Centrifugal Concentrators) and/or dilution with formulation buffer.
• centrifugation such as Arnicon Ultra 10,000 MWCO Centrifugal Concentrators
• compositions can be tested for long-term stability by size exclusion chromatography (SEC), denatured SEC (dSEC), hydrophobic interaction chromatography (HIC), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and for binding and bioactivlty at various timepoints.
• SEC size exclusion chromatography
• dSEC denatured SEC
• HIC hydrophobic interaction chromatography
• SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis
• bioactivity can be measured by any number of well-known assays.
• composition will be stable over the term of two years or more.
• STEP 1 Cell Expansion.
• cell expansion necessary to generate a sufficient number of cells for inoculation of a production bioreactor is performed using a clone of CHO celis expressing the etanercept fusion protein.
• the product of this expression process results in a mixture of correctly folded etanercept, as well as incorrectly folded and/or aggregated etanercept, along with additional impurities.
• the harvested cell culture fluid comprising such protein mixture is subjected to detergent viral inactivation.
• Affinity Chromatography is performed on the harvested cell culture obtain in Step 1 above using a conventional Protein A affinity column in a well known manner. Product recovery is approximately 85%.
• the product obtained is a complex protein mixture comprising correctl folded etanercept, incorrectly folded etanercept, and/or aggregates of correctly and/or Incorrectly folded etanercept, or protein fragments.
• the product obtained from this Protein A affinity column purification step is adjusted to pH 3,5 and then subjected to a viral inactivation step. Following viral inactivation the product is adjusted to pH 5.5 and then further clarified in a known manner using a commercially obtained capsule filter.
• the Protein A pool from step 2 above is diluted to ⁇ 8 mS/cm with WFI and applied to a column loading of up to 15 g/L media for each cycle.
• the column is operated at a linear velocity of 200 cm/h to give a 6 minute residence time.
• the column is washed with 2 CV of 25 mM acetate pH 5.5.
• the product is then eluted with an 8.5 CV, 15% to 85% gradient of 25 mM acetate pH 5.5 to 25 mM acetate, 0.7 U NaCi, pH 5.5.
• Product collection begins at 0.15 OD (A280, 1.0 cm path length) and collection ends at 50% of peak maximum.
• the eiuate volume is approximately 5 CV.
• STEP 3B Mixed Mode Anion Exchange Chromatopgraphy.
• a 27.0 L (45 cm diameter X 17 cm bed height) packed bed GE Healthcare Capto Adhere chromatography column is used to further purify the product obtained in step 3A above.
• the column Prior to use, the column is equilibrated with 2 CV of 25 mM Tris, pH 8.0 and sanitized with 2 CV/ of 0.1 N NaOH, 1M NaCI and neutralized and equilibrated with 2 CV of 25 mM Tris, pH 8.0.
• the Capto MMC pool from Step 3A above is adjusted to pH 8.1 with -0.045 kg of 1 M Tris, pH 8.3 per kg of pool.
• the product from Step 3A above was diluted in-line 1 :3.8 with F! to adjust the conductivity to 12.0 mS/cm and pH 8.0.
• the resulting material is then applied to a column loading of up to 15 g/L media.
• the column is operated at a linear velocity of 170 cm/h to give a 6 minute residence time. After loading, the column is washed with 2 CV of 25 mM Tris, pH 8.0.
• the product is then eluted with a 10 CV gradient (20% to 90%) of 25 mM Tris, pH 8.0 to 10 mM Tris, 1 M NaCI, pH 8.0.
• Product collection is started at 0.15 OD (A280, 1.0 cm path length) and collection ended at 25% of peak maximum.
• the eiuate volume is 4-6 CV.
• the eiuted product is filtered using a commercially available capsule filter and then subjected in a known manner to viral filtration and tangential flow filtration steps.
• Overall product recovery from step3B (including the final viral and tangential flow filtration steps) was approximately 68%.
• Product recovery measured before the filtration steps was about 75%.
• a schematic representation of HIC data obtained on eluation fractions from this step are representing in Figure 12.
• the final filtered product obtained in this example is found to have greater than about 90 wt % correctly folded etanercept as determined by HIC: less than 5 wt% incorrectly folded etanercept species as determined by HIC; less than about 3 wt% of clipped material by HIC analysis (beiieved to be fragments of etanercept in which the TNFR portion thereof has been truncated) and a combined amount of correctly and incorrectly folded etanercept of greater than 95 wt% as determined by size exclusion chromatography,
• samples of the etanercept formulations exemplified above were sterile filtered in a bio safety cabinet. Using sterilized pipettes and autoclaved pipette tips, sampies of the etanercept formulations were transferred to pre-labeled and autoclaved 1 mL lyophiiization vials. Vials were stoppered with sterile butyl stoppers and crimped with aluminum caps. All vials were then transferred to thermal stability ovens. Samples were subject to two thermal stability regimes: (1) two weeks at 40 and (2) four weeks at 25 °C. Throughout this specification, these two temperature regimes are denoted "T and T4," respectively.
• Etanercept formulations disclosed herein were analyzed using the well known technique of Size Exclusion Chromatography (SEC), a high-performance liquid chromatography method in which anaiytes are separated by size (see Rogner, , (2000). Size Exclusion Chromatography. Protein Liquid Chromatography. M. Kastner. Amsterdam, Elsevier. 61 : 89-145.).
• SEC Size Exclusion Chromatography
• the mobile phase buffer was prepared to contain 50 mM sodium phosphate monobasic monohydrate and 150 mM arginine. The pH was adjusted to 8.5 using 1 M HCI. All separations were performed using a Tosoh TSK-Gel SWxl 6 mm x 4 cm guard column (cat. no. 8543) attached linearly to a Tosoh TSK-Gel G4000 SWxl 7.8 mm x 30 cm (cat. no. 8542).
• the columns were brought to room temperature (23°C) and equilibrated with mobiie phase at a fiow rate of 0,5 mL/min. 5 microliters of 50 mg/mL etanercept formulation were injected onto the column using an autosamp!er. The separation was accomplished over 30 minutes at a flow rate of 0.5 mlJminute. Column eluent was monitored at a wavelength of 280 nm during this time.
• AH integration was performed using Chromeieon software (Dionex). Prior to integration, the SEC chromatogram for a buffer containing no etanercept was subtracted from all chromatograms. A!l integration was performed between retention times of 12 minutes and 26 minutes. Several parameters were used to define a peak. The minimum area for a detected peak was set to 0.05 mAu * min. The two- dimensional sensitivity for peak defection was set to 0.01 mAu and 75 seconds. Peak shoulders were added manually using a manual integration too!. Ail detected peaks were manually adjusted in two steps. First, peak baselines (the bottom boundary of the peak) were adjusted to horizontal. Secondly, the vertical positions of the peak baselines were adjusted to that of the chromatogram baseiine. The chromatogram baseiine value was defined as the signal in absence of ana!yte. The signal i absence of analyte was defined as the absorbance in mAu at 12 minutes retention time.
• the foiiowing tables shows the relative amounts of Aggregates, Monomer and Fragment 3 determined by SEC analysis as described above.
• T 0 formulation maintained at 5 C and analyzed within 24 hours of creation.
• HSC chromatography may be carried out in a manner known in the art and generally described in U.S. Patent 7,294,481 , incorporated herein by reference, Samples are evaluated at t 0 (within 24 hours of preparation at 5°C.) and again after either two weeks of storage at 25°C. (t 2 ) or after 4 weeks of storage at 25°C.
• Peak 1 in the H!C chro.matogram is believed to be or include "Fragment 3", which is identified and quantified using SEC, as referenced above in the discussion of SEC data; Peak 2 is etanercept monomer as referenced above in the discussion of SEC data; and Peak 3 includes "Aggregate(s)” as referenced above in the discussion of SEC data.
• Peak 1 peak 1
• peak 2 peak 2
• peak 3 includes "Aggregate(s)” as referenced above in the discussion of SEC data.

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